Gene expression in eukaryotes is induced at a region of a DNA sequence referred to as a promoter. Generally, a promoter is located upstream of codon regions, has a binding site for RNA polymerase, and controls the transcriptional initiation of DNA located downstream thereof. The promoter region also contains other elements functioning as regulatory genes for gene expression. The promoter region has a TATA box consensus sequence at position approximately −30 bp on the 5′ end from the initiation codon and often has a CAAT box consensus sequence at position approximately −75 bp from the same (Breathnach and Chambon (1981) Ann. Rev. Biochem. 50: 349–383 (non-patent document 1); Messing et al., Genetic Engineering of Plants, T. Kosuge, Meredish and Hollaender (ed), pp. 211–227 (1983) (non-patent document 2)).
In plants, the CAAT box may be substituted with a consensus sequence located at the same distance as that of the CAAT box from a cap site, which is named the AGGA box by Messing et al. (non-patent document 2). Transcription is initiated when a transcriptional factor (protein) binds to the TATA box consensus sequence. Next, at the signal of this binding, RNA polymerase and other transcriptional factors bind, initiating transcription from the transcription initiation point. When RNA polymerase reaches a terminator indicating transcription termination, transcription is completed. A promoter is a binding site for RNA polymerase and also regulates the direction in which an enzyme moves over DNA. Hence, which one of the double strands becomes a template strand is determined depending on the position of a promoter. Efficient expression of a gene introduced into a plant cell is an important issue in the production of transgenic plants. A promoter sequence is a major factor in determining the transcriptional level of a gene within a plant cell. Generally, the use of a promoter sequence with strong transcriptional activity enables improvement of the expression level of a target gene. It is known that in promoter sequences, there are specificities based on differences in RNA polymerases that differ depending on plant species, organs, tissues, cells, or environmental conditions. Therefore, to carry out gene expression in a plant cell, it is necessary to use a promoter sequence that functions under purpose conditions.
Conventionally, examples of promoters capable of functioning within plants include a 35S promoter (EP0131623 (patent document 1)) derived from a cauliflower mosaic virus (CaMV), promoters (nopaline synthase (nos) and mannopine synthase (mas)) that can be found in Agrobacterium T-DNA, and an octopine synthase (ocs) promoter (EP0122791 (patent document 2), EP0126546 (patent document 3), and EP0145338 (patent document 4)).
However, it is known that dicotyledons and monocotyledons differ in their transcription efficiency. It has been reported that although CaMV-derived 35S promoter is a strong promoter that causes high-level RNA production in a wide variety of types of plants, including plants that are far from the viral host range, the 35S promoter has only relatively low activity in agriculturally important gramineous plants (Shimamoto et al., 1989 Nature 338, 274–276 (non-patent document 3); Rhodes et al., 1988 Science 240, 204–207 (non-patent document 4)). In a promoter of a monocotyledon, introns are present in the 5′ untranslation region of a gene to be induced thereby, and these are absent in dicotyldons and are necessary for activating thereby, and these are absent in dicotyldons and are necessary for activating gene expression. Hence, it is often impossible to obtain effective expression levels by simple ligation thereof. Therefore, in monocotyledons, improvement of the 5′ untranslation region, use of introns, and gene resynthesis have been devised to increase expression levels (Plant Mol. Biol. 32 (1996) 393–405 (non-patent document 5); Plant Cell Rep. 6: 265–270 (non-patent document 6)). According to other studies, Kay et al., (Science 236: 1299–1302 (1987) (non-patent document 7)) have reported transcriptional activity that is ten times higher in tobacco plants that had experienced gene transfer and that had a CaMV-derived 35S promoter. They have also reported that the CaMV-derived 35S promoter upstream sequence of 260 bp was contained twice.
Furthermore, Ow et al., (Proc. Natl. Acad. Sci. 84: 4870–4874 (1987) (non-patent document 8)) have reported that multimeric structure of the terminal region of a 35S promoter (between positions −148 and −89) can activate a 35S promoter core to an expression level higher than that obtained by a natural 35S promoter. Furthermore, Fang et al., (The Plant Cell 1: 141–150 (1989) (non-patent document 9)) have reported that monomeric structure and multimeric structure of upstream 35S promoter fragments (between −209 and −46) can act as enhancers to increase transcription from heterologous promoters. In these studies, 8 repeats of the upstream region between positions −209 and −46 of a 35S promoter have been cloned at position −50 of an rbcS-3A gene, which is a small subunit of ribulosebisphosphate carboxylase. The octamer increased rbcS-3A transcription to levels higher than those obtained using an rbcS-3A upstream region (non-patent document 9).
Under such circumstances, it is extremely important to provide a promoter having strong activity for molecular breeding of plants using transformation methods. The importance of a promoter that functions efficiently, particularly in monocotyledons to which major grains such as rice, wheat, and corn belong, is particularly high. Hence, as promoters derived from monocotyledons, the use of several examples including a rice actin promoter (U.S. Pat. No. 5,641,876 (patent document 5)) and a corn ubiquitin promoter (U.S. Pat. No. 5,510,474 (patent document 6)) has been attempted.
In addition, acetolactate synthase (ALS) is a common enzyme at the initial stage of the branched amino acid biosynthetic pathway that is present in plants and microorganisms. It is known as a target enzyme of at least four structurally different herbicides (including sulfonylureas, imidazolinones, triazolopyrimidinesulfonamides, and pyrimidinecarboxy herbicides). In addition, acetolactate synthase (ALS) is not present in animals, so that these herbicides have a slight effect on human bodies. Moreover, the nucleotide sequence of ALS gene is highly conserved, particularly in plants (Mazur et al., Annu Rev Plant Physiol 40 441–447 (1987) (non-patent document 10)). Gail et al. (Pesticide Sci. 30(4) 418–419 (1990) (non-patent document 11)) have examined expression of acetolactate synthase (ALS) in various tissues including leaves, stems, roots, flowers, pods, and meristems by enzyme assay using Lima beans. ALS activity was measured on days 14, 28, and 42 after sowing in leaves, stems, roots, and meristems, and ALS activity was measured on day 42 after sowing in flowers and pods. Gail et al. have reported that the activity decreased with aging in tissues other than stems, but ALS was expressed constitutively in any tissue. Sharon J. Keeler et al. have observed the constitutive expression of ALS in various tobacco tissues including seedlings, leaves, stems, roots, and flowers (Plant Physiol. 102, 1009–1018 (1993) (non-patent document 12)). ALS expression was shown to be highest in developing organs such as seedlings and young leaves and lowest in mature leaves. Also, in the results of in situ hybridization, the highest ALS expression was observed in metabolically active cells of roots, stems, and flowers or dividing cells. For example, in roots, expression was highest in the tips of the roots. The further the distance from the tips (that is, the older the cells), the gradually lower the expression. Regarding the transcription products of ALS, expression of approximately 0.1% of all mRNA was observed and a maximum 4-fold difference was observed in expression levels among tissues.    [Patent Document 1] EP 0131623    [Patent Document 2] EP 0122791    [Patent Document 3] EP 0126546    [Patent Document 4] EP 0145338    [Patent Document 5] U.S. Pat No. 5,641,876    [Patent Document 6] U.S. Pat. No. 5,510,474    [Non-patent Document 1] Breathnach and Chambon (1981) Ann. Rev. Biochem. 50: 349–383    [Non-patent Document 2] Messing et al., Genetic Engineering of Plants, T. Kosuge, Meredish and Hollaender (ed), pp. 211–227 (1983)    [Non-patent Document 3] Shimamoto et al., 1989 Nature 338, 274–276    [Non-patent Document 4] Rhodes et al., 1988 Science 240, 204–207    [Non-patent Document 5] Plant Mol. Biol. 32 (1996) 393–405    [Non-patent Document 6] Plant Cell Rep. 6: 265–270    [Non-patent Document 7] Science 236: 1299–1302 (1987)    [Non-patent Document 8] Proc. Natl. Acad. Sci. 84: 4870–4874 (1987)    [Non-patent Document 9] The Plant Cell 1: 141–150 (1989)    [Non-patent Document 10] Mazur et al., Annu Rev Plant Physiol 40 441–447    [Non-patent Document 11] Pesticide Sci. 30(4) 418–419 (1990)    [Non-patent Document 12] Plant Physiol. 102, 1009–1018 (1993)